Project Summary/Abstract This is a revised competitive renewal of NIH grant DK055545 which is focused on the role of phosphatidylinositol 3-kinase (PI3K) in insulin action and insulin resistance. PI 3-kinase is a critical node in insulin's metabolic actions. Alterations in PI3K have been implicated in cancer, diabetes and many other disorders. In previous work under this grant we have used both in vitro and in vivo approaches to define the role of this enzyme in insulin action and insulin resistance. We have shown that regulation of PI3K depends both on the nature of the different regulatory subunits; the stoichiometry between regulatory and catalytic subunits; the ability of PI 3-kinase to serve as a site for divergence of downstream signaling; and alterations in PI3K activity in disease states. We have also identified new links between the PI 3-kinase pathway and other signaling pathways, including important links between the p85 regulatory subunits and several pathways involved in insulin resistance, such as activation of the stress kinases JNK and p38, regulation of the PIP3 phosphatase PTEN, and a novel link between PI 3-kinase, endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) created by the interaction between p85? and XBP-1s, facilitating XBP-1s transport into the nucleus and thus modifying the ER stress response. Another exciting recent development has been the identification of a mutation in p85? in patients with SHORT syndrome, a syndrome characterized by insulin resistance and partial lipodystrophy. Recently, we have created a knock-in mouse bearing this mutation to study its effects in vivo. We have also begun to characterize the different roles of the two major catalytic subunits of PI3K (p110? and p110?) in insulin signaling and mitochondrial homeostasis through knockout in vivo and in vitro. This has led to new hypotheses about the unique roles of the different catalytic and regulatory subunits of PI 3-kinase, which allow these proteins to serve as both sites of divergence in the insulin signaling pathway and sites of positive and negative regulation in physiological and pathological states. In the next five years, we propose to expand upon these observations by defining at both the molecular and physiological levels how different signals are generated by the p110? and p110? catalytic subunits of PI 3- kinase, the specific signaling complexes involved, and the link between PI3K and mitochondrial homeostasis. In addition, we will expand our studies on the regulatory subunits focusing defining the regions of p85? that interact with XBP-1s creating crosstalk between the PI 3-kinase pathway and ER stress. We will also further define how mutations in the p85? regulatory subunit can have a dominant negative effect and result in severe insulin resistance. Together, these studies will help complete our understanding of the role of the PI3K system and its different catalytic and regulatory subunits in insulin action and insulin resistance.